| Assay Method Information | |
| | Determination of EC50 Values for 5-HT4 Receptor |
| Description: | A stable CHO cell line expressing recombinant human 5-HT4 receptor and pCRE-Luc reporter system was used for cell-based assay. The assay offers a non-radioactive based approach to determine binding of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP, which is modulated by activation, or inhibition of the receptor is measured. The recombinant cells harbor luciferase reporter gene under the control of cAMP response element. The above cells were grown in 96 well clear bottom white plates in Ham's F12 medium containing 10% fetal bovine serum. Prior to the addition of compounds or standard agonist, cells were serum starved overnight. Increasing concentrations of test compounds were added to the cells in OptiMEM medium. The incubation was continued in CO2 incubator at 37° C. with 5% CO2 conditions for 4 h. Medium was removed and cells were washed with phosphate buffered saline. The cells were lysed and luciferase activity was measured in a Luminometer. Concentration-response data was generated using the luciferase assay, following incubation of cells with receptor ligands, were analyzed by subtracting basal levels (i.e. with medium alone), then normalizing values as a percentage of controls (endogenous agonist serotonin (10 μM)). This data were analyzed by nonlinear regression with variable slope, using the computer package GraphPad Prism 4. EC50 values of the compounds were defined as the concentration required in stimulating the luciferase activity by 50%. |
| Affinity data for this assay | |
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